Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase. ADMA accumulation, mainly due to a decreased dimethylarginine dimethylaminohydrolase (DDAH) activity, has been related to the development of cardiovascular diseases. We investigate whether estradiol prevents the alterations induced by oxidized low density lipoprotein (oxLDL) on the DDAH/ADMA/NO pathway in human umbilical artery endothelial cells (HUAEC). HUAEC were exposed to estradiol, native LDL (nLDL), oxLDL and their combinations for 24h. In some experiments, cells were also exposed to the unspecific estrogen receptor (ER) antagonist ICI 182780, or the specific ERα antagonist MPP. ADMA concentration was measured by HPLC and concentration of NO by amperometry. Protein expression and DDAH activity were measured by immunoblotting and an enzymatic method, respectively. oxLDL, but not to nLDL, increased ADMA concentration with a concomitant decrease of DDAH activity. oxLDL reduced eNOS protein and NO production. Estradiol alone had no effects on DDAH/ADMA/NO pathway, but increased the attenuated endothelial NO production induced by oxLDL by restoring the DDAH activity. ICI 182780 and MPP completely abolished these effects of estradiol on oxLDL-exposed cells. In conclusion, estradiol restores DDAH activity ADMA levels and NO production impaired by oxLDL in HUAEC acting through ERα.